Elsevier

NeuroImage

Volume 26, Issue 1, 15 May 2005, Pages 132-140
NeuroImage

Demyelination increases radial diffusivity in corpus callosum of mouse brain

https://doi.org/10.1016/j.neuroimage.2005.01.028Get rights and content

Abstract

Myelin damage, as seen in multiple sclerosis (MS) and other demyelinating diseases, impairs axonal conduction and can also be associated with axonal degeneration. Accurate assessments of these conditions may be highly beneficial in evaluating and selecting therapeutic strategies for patient management. Recently, an analytical approach examining diffusion tensor imaging (DTI) derived parameters has been proposed to assess the extent of axonal damage, demyelination, or both. The current study uses the well-characterized cuprizone model of experimental demyelination and remyelination of corpus callosum in mouse brain to evaluate the ability of DTI parameters to detect the progression of myelin degeneration and regeneration. Our results demonstrate that the extent of increased radial diffusivity reflects the severity of demyelination in corpus callosum of mouse brain affected by cuprizone treatment. Subsequently, radial diffusivity decreases with the progression of remyelination. Furthermore, radial diffusivity changes were specific to the time course of changes in myelin integrity as distinct from axonal injury, which was detected by βAPP immunostaining and shown to be most extensive prior to demyelination. Radial diffusivity offers a specific assessment of demyelination and remyelination, as distinct from acute axonal damage.

Introduction

Diverse neurological disorders involve white matter pathology leading to myelin and axonal dysfunction (Cammer and Zhang, 1999, De Stefano et al., 2001, McGavern et al., 1999, Perry and Anthony, 1999, Trapp et al., 1999, van der Valk and De Groot, 2000). Accurate assessments of these conditions may be highly beneficial in evaluating and selecting therapeutic strategies for patient management. However, neurological examination may not distinguish primary myelin damage from injury that involves axonal pathology. Thus, the development of a non-invasive biological marker that is capable of detecting and differentiating axonal and myelin damage would have significant clinical implications.

Magnetic resonance imaging (MRI) is a well-established method that is widely applied clinically for detecting various central nervous system (CNS) injuries and diseases (Arfanakis et al., 2002, De Stefano et al., 2002, Parkinson et al., 2002). However, conventional MRI techniques, such as T1- and T2-based measurements, are not capable of differentiating axonal versus myelin pathology in CNS white matter disorders. For example, it has been reported that axonal injury in spinal cord of multiple sclerosis patients is independent of T2 lesions (Bergers et al., 2002). In addition, there was no difference in T2W images between brains of WT and dysmyelinated shiverer mice, i.e., 100% myelinated tracts vs. tracts without myelin sheath, in our previous study (Song et al., 2002). Recently, an analytical approach examining diffusion tensor imaging (DTI) derived parameters has been proposed to assess the extent of axonal damage, demyelination, or both (Song et al., 2002, Song et al., 2003).

The non-invasive nature and high sensitivity of DTI to microscopic structural tissue changes has resulted in its wide application in developmental and pathological examinations of CNS in both animal models and humans (Filippi et al., 2001, Ito et al., 2001, Maldjian and Grossman, 2001, Nusbaum et al., 2001). After generation of the diffusion tensor matrix from a series of diffusion weighted images, the three eigenvalues or diffusivities (λ1, λ2, and λ3) are calculated by matrix diagonalization (Basser and Pierpaoli, 1998, Pierpaoli and Basser, 1996). The basis vectors of a local frame of reference coincide with the eigenvectors of the diffusion tensor. This local frame reflects white matter tract directionality at the voxel level. In the CNS white matter, λ1 represents the water diffusivity parallel to the axonal fibers and is referred to as λ||, the axial diffusivity. The water diffusivities perpendicular to the axonal fibers, λ2 and λ3, are averaged and referred to as λ = (λ2 + λ3)/2, the radial diffusivity (Song et al., 2002, Song et al., 2003). Previous studies from these laboratories have examined the potential for λ and λ|| to differentially detect myelin and axonal abnormalities in shiverer mice (Song et al., 2002) and optic nerve from a mouse model of retinal ischemia (Song et al., 2003). Specifically, our previous results demonstrate that axonal damage leads to a marked decrease in λ|| and modest–often insignificant–decreases in λ while demyelination increases λ without changing λ||. These DTI results were further validated with immunohistochemistry analyses supporting the hypothesis that λ|| and λ hold promise as specific markers of axonal and myelin damage, respectively.

To demonstrate that λ can distinguish myelin damage relative to axonal damage in a reproducible model of primary demyelination relevant to multiple sclerosis, the current study used the cuprizone model that exhibits consistent demyelination of corpus callosum (CC) in mouse brains (Armstrong et al., 2002, Matsushima and Morell, 2001, Stidworthy et al., 2003). A reproducible time course of oligodendrocyte loss and subsequent demyelination in the corpus callosum is observed after feeding 0.2% cuprizone to C57BL/6 male mice starting at 8 weeks of age. Cuprizone is a neurotoxicant that chelates copper, which is essential for proper function of many metalloenzymes. Demyelination is extensive after several weeks of dietary cuprizone, yet can be reversed after returning the mice to normal chow. Previous reports suggested that in multiple sclerosis and other experimental models of demyelination (Kuhlmann et al., 2002, Onuki et al., 2001), the most extensive axonal damage occurs during the early phase of demyelination and macrophage/microglial reaction, thus further examination of axonal integrity of CC has also been performed in the current study.

The reproducible demyelination and remyelination of CC are features that make the cuprizone model an excellent test bed for the current hypothesis that increased radial diffusivity (λ) may be used as a biological marker of white matter demyelination. In addition to CC, anatomy-based region of interest (ROI) analyses were performed on six additional mouse brain white matter tracts including anterior commissure (AC), cingulum (Cg), cerebral peduncle (CP), external capsule (EC), optic tract (OT), and superior cerebellar peduncle (SCP) to demonstrate the capability of DTI to analyze multiple tracts in a single experimental setting with the mouse as a valuable laboratory subject. However, consistent demyelination from cuprizone intoxication has been observed/validated only for CC in mouse brain. Thus, for validation/testing of our hypothesis, only CC will be analyzed using both DTI and histology. Our DTI results indicate that the time course of increased radial diffusion in CC occurs in parallel with the histological evidence of loss of myelin integrity.

Section snippets

Animals

Mice were maintained in the USUHS animal housing facility and all procedures were performed in accordance with guidelines of the National Institutes of Health, the USUHS Institutional Animal Care and Use Committee, and the Society for Neuroscience.

Cuprizone treatments

Eight-week-old male C57BL/6 mice (Jackson Labs) were placed on a diet of 0.2% (w/w) cuprizone [finely powdered oxalic bis(cyclohexylidenehydrazide); Aldrich, Milwaukee, WI] thoroughly mixed into milled chow (Harlan Teklad, Madison, WI), which was

Volume-averaged DTI parameters

Mouse brain white matter tracts are readily identified in relative anisotropy (RA) maps (Fig. 1). The RA maps illustrate that cuprizone ingestion differentially alters signal from specific white matter tracts, as is consistent with previous histological reports of myelin integrity (Matsushima and Morell, 2001, Stidworthy et al., 2003). For example, the reduction of RA in cingulum (Cg), corpus callosum (CC), and external capsule (EC) after cuprizone treatment is apparent as reduced intensity in

Discussion

The capability of DTI parameters to detect the extent of demyelination and spontaneous remyelination in specific CNS regions was assessed in a well-characterized model of experimental demyelination. Extensive demyelination of the CC has been consistently reported in C57BL/6 mice treated with 0.2% cuprizone (Hiremath et al., 1998, Mason et al., 2001, Matsushima and Morell, 2001). Also, the time course of disease progression in the CC is relatively reproducible (Armstrong et al., 2002, Matsushima

Acknowledgments

This study was supported in part by the National Multiple Sclerosis Society (RG 3376-A-2/1, and CA 1012-A-13), the Washington University Alzheimer's Disease Research Center (NIH: AG05681), the Washington University Small Animal Imaging Resource (WUSAIR) (NIH: R24-CA83060), and the National Institutes of Health (NS39293). Helpful discussions with Dr. Christopher D. Kroenke and members of the Washington University Biomedical MR Laboratory are gratefully acknowledged. We thank Sue Pletcher for

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